Hydroporphyrins for photoacoustic imaging

ABSTRACT

Provided are photoacoustic imaging contrast agents that include at least one radiation-absorbing component comprising a bacteriochlorin, a metallobacteriochlorin, a derivative thereof, or a combination thereof. Also provided are methods for using the disclosed photoacoustic imaging contrast agents either singly or in combination for generating an image of a volume, optionally a subject or a body part, cell, tissue, or organ thereof. Further provided are compositions and methods for multiplex photoacoustic imaging of a volume, optionally a subject or a body part, cell, tissue, or organ thereof using photoacoustic imaging contrast agents that include a plurality of the presently disclosed bacteriochlorins, metallobacteriochlorins, and/or derivatives thereof simultaneously.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/454,493, filed Feb. 3, 2017, the disclosure ofwhich is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The presently disclosed subject matter relates generally tohydroporphyrins and hydroporphyrin conjugates, chelates, andderivatives, and methods for using the same as contrast agents forphotoacoustic imaging. In particular, the presently disclosed subjectmatter relates to metallobacteriochlorins and methods of using the samein photoacoustic imaging methodologies.

BACKGROUND

Photoacoustic Imaging (PAI) is an emerging medical imaging modality thatis based on the phenomenon of conversion of optical energy into acousticenergy (Bell, 1880). PAI offers distinct advantages over other strictlyoptical imaging methods such as fluorescence because physiologicaltissue poses considerably less interference for acoustic waves than itdoes for light. Although PAI by definition still requires opticalexcitation and subsequent loss of light through endogenous absorptionand scattering, the lower interference of the acoustic signal responseallows imaging of features at much greater depths, up to 5 or even 7 cm(de Zerda et al., 2012; Wilson et al., 2013; Wang & Yao, 2016).

In addition to collecting signals from endogenous biomolecules such asmelanin and hemoglobin, PAI can be used to detect both general contrastagents and targeted PAI probes. Typically, such reagents have their peakoptical absorption within the near infrared (NIR) spectral region (e.g.,680-1100 nm), where biological interference is reduced. Although a fewNIR reagents are in development such as IRDye 800CW (Marshall et al.,2010), quantum dots, gold nanoparticles, and carbon nanotubes (de Zerdaet al., 2012) most PAI studies to date have employed either endogenousprobes (e.g., melanin or hemoglobin) or the exogenous probe IndocyanineGreen (ICG), a carbocyanine dye which has received regulatory approvalbut suffers from a significantly broad absorption spectrum (see FIG. 1).ICG's broad absorption spectrum leads to a corresponding broad PAIsignal that limits the number of additional targeted biomarkers that canbe distinguished in a typical experiment when ICG is employed as thecontrast agent.

Ideally, clinicians would like to measure multiple biomarkers in asingle run for confirmation of complex diseases such as cancer. To makethe simultaneous detection of multiple biomarkers within the NIRspectral range possible, disclosed herein are PAI reagents based onsynthetic bacteriochlorins, a class of PAI agents that offer extremelynarrow absorption and PAI spectra, thereby enabling multiplex detectionwith minimal overlap and within a compact NIR spectral window (e.g.,650-1070 nm).

SUMMARY

This Summary lists several embodiments of the presently disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

The presently disclosed subject matter provides in some embodimentsphotoacoustic imaging (PAI) contrast agents. In some embodiments, thePAI contrast agents comprise at least one radiation-absorbing componentthat comprises a bacteriochlorin, a metallobacteriochlorin, a derivativethereof, or a combination thereof. In some embodiments, the PAI contrastagent comprises a plurality of different bacteriochlorins,metallobacteriochlorins, derivatives thereof, or combinations thereof,each bacteriochlorin, metallobacteriochlorin, or derivative having adifferent absorption spectrum in the range of 650-1070 nm. In someembodiments, the photoacoustic imaging contrast agent comprises at leastthree different bacteriochlorins, metallobacteriochlorins, orderivatives thereof, wherein each bacteriochlorin,metallobacteriochlorin, or derivative thereof has an absorption spectrumwith a peak absorption value in the range of 700-950 nm; and the atleast three absorption spectra are substantially non-overlapping in therange of 700-950 nm. In some embodiments, the metallobacteriochlorincomprises a metal selected from the group consisting of nickel, iron,and cobalt.

The presently disclosed subject matter also provides in some embodimentsmethods for generating an image of a volume or a part thereof. In someembodiments, the methods comprise administering to the volume or thepart thereof a contrast agent comprising at least oneradiation-absorbing component comprising a bacteriochlorin, ametallobacteriochlorin, a derivative thereof, or a combination thereofexposing the volume or the part thereof to radiation; detectingultrasonic waves generated in the volume or the part thereof by theradiation; and generating a photoacoustic image therefrom of the volumeor the part thereof containing the administered contrast agent. In someembodiments, the bacteriochlorin, the metallobacteriochlorin, or thederivative thereof is a component of and/or encapsulated in a micelle, aliposome, a nanoparticle, or a combination thereof. In some embodiments,radiation with a wavelength of 650-1070 nm is used. In some embodiments,radiation with a wavelength of 650-900 nm, 700-950 nm, and/or 750-950 nmis used. In some embodiments, the physiologically tolerable contrastagent comprises a plurality of different bacteriochlorins,metallobacteriochlorins, derivatives thereof, or combinations thereof,each bacteriochlorin, metallobacteriochlorin, or derivative having adifferent absorption spectrum in the range of 650-1070 nm. In someembodiments, the contrast agent comprises a targeting agent. In someembodiments, the targeting agent comprises a moiety that binds to aligand and/or a target present on a tumor cell or a cancer cell, or avascular endothelial cell associated therewith. In some embodiments, theligand and/or a target comprises a tumor-associated antigen. In someembodiments, the moiety comprises a peptide or peptide mimetic thatbinds to a tumor-associated antigen.

The presently disclosed subject matter also provides in some embodimentsmethods for multiplex photoacoustic imaging of a volume or a partthereof. In some embodiments, the methods comprise administering to thevolume or the part thereof a contrast agent comprising a plurality ofradiation-absorbing components, each member of the plurality ofradiation-absorbing components comprising a bacteriochlorin, ametallobacteriochlorin, a derivative thereof, or a combination thereofexposing the volume or a part thereof to radiation, wherein theradiation is calibrated to wavelengths that are differentially absorbedby the plurality of radiation-absorbing components; differentiallydetecting ultrasonic waves generated in the volume or the part thereofby the radiation as it is differentially absorbed by the plurality ofradiation-absorbing components; and generating a photoacoustic imagetherefrom of the volume or the part thereof containing the administeredcontrast agent, wherein the photoacoustic image is generated from thedifferentially detecting ultrasonic waves. In some embodiments, one ormore of the plurality of the bacteriochlorins, metallobacteriochlorins,or derivatives thereof is a component of and/or encapsulated in amicelle, a liposome, a nanoparticle, or a combination thereof. In someembodiments, radiation with a wavelength of 650-1070 nm is used. In someembodiments, radiation with a wavelength of 650-900 nm, 700-950 nm,and/or 750- 950 nm is used. In some embodiments, each member of theplurality of radiation-absorbing components has a different absorptionspectrum in the range of 650-1070 nm. In some embodiments, one or moreof the members of the plurality of radiation-absorbing componentscomprises a targeting agent. In some embodiments, the targeting agentcomprises a moiety that binds to a ligand and/or a target present on atumor cell or a cancer cell, or a vascular endothelial cell associatedtherewith. In some embodiments, the ligand and/or a target comprises atumor-associated antigen. In some embodiments, the moiety comprises apeptide or peptide mimetic that binds to a tumor-associated antigen. Insome embodiments, two or more of the members of the plurality ofradiation-absorbing components comprise a targeting agent. In someembodiments, the two or more of the members of the plurality ofradiation-absorbing components comprise different targeting agents. Insome embodiments, the different targeting agents bind to and/orotherwise accumulate in the same or different targets and/or targetedsites.

In some embodiments of the methods of the presently disclosed subjectmatter, the volume is a subject or a body part thereof, optionally acell, tissue, and/or organ thereof. In some embodiments, the volumecomprises a tumor cell, a cancer cell, or a tumor- or cancer-associatedvascular cell. In some embodiments, the contrast agent is aphysiologically tolerable contrast agent or a plurality ofphysiologically tolerable contrast agents. In some embodiments, thecontrast agent is physiologically tolerable for use in a human. In someembodiments, the contrast agent is provided in a pharmaceuticalcomposition comprising the photoacoustic imaging contrast agent and apharmaceutically acceptable carrier, diluent, or excipient. In someembodiments, the pharmaceutical composition is pharmaceuticallyacceptable for use in a human. In some embodiments, the volume comprisesone or more targets and/or targeted sites that can be targeted by atargeting agent.

In some embodiments, the presently disclosed subject matter alsoprovides photoacoustic imaging contrast agents. In some embodiments aphotoacoustic imaging contrast agent of the presently disclosed subjectmatter comprises at least one radiation-absorbing component comprising abacteriochlorin, a metallobacteriochlorin, a derivative thereof, or acombination thereof. In some embodiments, the at least oneradiation-absorbing component comprises a compound selected from thegroup consisting of:

In some embodiments, the at least one radiation-absorbing componentcomprises a derivative of B1-B3 and B107 comprising a complexed metal,wherein the complexed metal is selected from the group consisting ofnickel, cobalt, and iron. In some embodiments, the derivative comprisesa compound selected from the group consisting of:

wherein M is a metal, optionally a metal selected from the groupconsisting of nickel, cobalt, and iron. In some embodiments, thephotoacoustic imaging contrast agent is physiologically tolerable foruse in a subject, optionally a human.

In some embodiments, the presently disclosed subject matter providespharmaceutical compositions. In some embodiments, the presentlydisclosed pharmaceutical compositions comprise one or more photoacousticimaging contrast agents as described herein and a pharmaceuticallyacceptable carrier, diluent, or excipient. In some embodiments, thepharmaceutical composition is pharmaceutically acceptable for use in ahuman.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plot of absorption spectra for ICG in water (adapted fromLandsman et al., 1976) vs. a panel of three bacteriochlorins in toluene.In this example, the spectrum for B2 (2,12-diphenyl) is represented bythat of the similar 2,12-dimesityl bacteriochlorin (from Chen et al.,2012).

FIGS. 2A-2C are the structures of bacteriochlorophyll a (FIG. 2A) ageneric design of stable wavelength-tunable tunable bacteriochlorins(FIG. 2B), and nickel-bacteriochlorin B107 (FIG. 2C).

FIGS. 3A and 3B are plots comparing ICG (dashes) andNi-metallobacteriochlorin B107 (solid line) signals imaged in agarphantoms at a depth of 3 mm (FIG. 3A) or 6 mm (FIG. 3B). Samples wereintroduced at equal optical density (7.5 OD) at the respective dyemaxima (795 and 765 nm).

FIG. 4 is a plot comparing B107 (black) versus ICG (gray) PAI signalintensity over time with laser at 800 nm.

FIG. 5 shows the structures of three bacteriochlorins (bacteriochlorinsB1-B3) with spectrally distinct absorption bands. Representativeabsorption spectra for bacteriochlorins B1-B3 are depicted in FIG. 1.

FIG. 6 depicts an exemplary non-limiting synthesis scheme forbacteriochlorins (B1: R¹═R²═H; B2: R¹═Phenyl, R²═CO₂Me; B3: R¹═Br andthen is converted to phenylethynyl groups through Sonogashira couplingat latter stage, R²═CO₂Me).

FIG. 7 shows examples of water soluble metallobacteriochlorins (left)and metallochlorins (right) with bioconjugatable tethers (M═Ni, Fe, Co).The carboxylates can be converted to a reactive ester for bioconjugationto amines on biomolecules (examples include N-hydroxy-succinimidyl,N-hydroxy-sulfo-succinimidyl, pentafluorophenyl, etc.) or to othergroups such as iodoacetamide or maleimides for coupling to thiols onbiomolecules.

FIG. 8 shows the structures of three metallobacteriochlorins(metallobacteriochlorins MB1-MB3), where M is a metal, optionally ametal selected from the group consisting of nickel, cobalt, and iron.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter, in which some, but not all embodiments of the presentlydisclosed subject matter are described. Indeed, the presently disclosedsubject matter can be embodied in many different forms and should not beconstrued as limited to the embodiments set forth herein; rather, theseembodiments are provided so that this disclosure will satisfy applicablelegal requirements.

I. Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the presentlydisclosed subject matter.

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. References to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques that would be apparent to one of skill in the art.While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

In describing the presently disclosed subject matter, it will beunderstood that a number of techniques and steps are disclosed. Each ofthese has individual benefit and each can also be used in conjunctionwith one or more, or in some cases all, of the other disclosedtechniques.

Accordingly, for the sake of clarity, this description will refrain fromrepeating every possible combination of the individual steps in anunnecessary fashion. Nevertheless, the specification and the claimsshould be read with the understanding that such combinations areentirely within the scope of the presently disclosed subject matter andclaims.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a PAI contrast agent”includes a plurality of PAI contrast agents, and so forth.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration, or percentage ismeant to encompass variations of in some embodiments, ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, andin some embodiments ±0.1% from the specified amount, as such variationsare appropriate to perform the disclosed methods.

As used herein, the term “and/or” when used in the context of a listingof entities, refers to the entities being present singly or incombination. Thus, for example, the phrase “A, B, C, and/or D” includesA, B, C, and D individually, but also includes any and all combinationsand subcombinations of A, B, C, and D.

The term “comprising”, which is synonymous with “including,”“containing,” or “characterized by” is inclusive or open-ended and doesnot exclude additional, unrecited elements or method steps. “Comprising”is a term of art used in claim language which means that the namedelements are present, but other elements can be added and still form aconstruct or method within the scope of the claim.

As used herein, the phrase “consisting of” excludes any element, step,or ingredient not specified in the claim. When the phrase “consists of”appears in a clause of the body of a claim, rather than immediatelyfollowing the preamble, it limits only the element set forth in thatclause; other elements are not excluded from the claim as a whole.

As used herein, the phrase “consisting essentially of” limits the scopeof a claim to the specified materials or steps, plus those that do notmaterially affect the basic and novel characteristic(s) of the recitedsubject matter.

With respect to the terms “comprising”, “consisting of”, and “consistingessentially of”, where one of these three terms is used herein, thepresently disclosed subject matter can include the use of either of theother two terms. For example, it is understood that the scopeencompassed by “comprising” can in some embodiments be broader than thatencompassed by “consisting essentially of”, which in some embodimentscan have a scope that is broader than “consisting of”. As such, it isfurther understood that the use of the term “comprising” alsoencompasses “consisting essentially of” as well as “consisting of”, andthe use of “consisting essentially of” also encompasses “consisting of”.

As used herein, the term “bacteriochlorin” refers to a largeheterocyclic aromatic ring consisting, at the core, of two pyrroles andtwo pyrrolines coupled through four ═CH— linkages. As used herein, theterm “bacteriochlorin” encompasses both bacteriochlorins andisobacteriochlorins, as well as derivatives including, but not limitedto metalated derivatives.

As used herein, the phrase “photoacoustic imaging contrast agent” refersto a composition that when contacted with a target (optionally a targetpresent within a subject) allows the target to be imaged byphotoacoustic imaging. In some embodiments, a photoacoustic imagingcontrast agent comprises at least one radiation-absorbing molecule,which in some embodiments comprises a bacteriochlorin, ametallobacteriochlorin, a derivative thereof, or a combination thereof.It is noted that a radiation-absorbing molecule can itself be aphotoacoustic imaging contrast agent. Thus, in those embodiments whereina combination of different radiation-absorbing molecules are present,the composition as a whole can be considered a photoacoustic imagingcontrast agent and, in some embodiments, each individualradiation-absorbing molecule can be considered a photoacoustic imagingcontrast agent.

As used herein, the phrase “substantially non-overlapping” as it relatesto absorption spectra means that the percent overlap of the absorptionspectra being compared is in some embodiments less than 50%, in someembodiments less than 40%, in some embodiments less than 30%, in someembodiments less than 25%, in some embodiments less than 20%, in someembodiments less than 15%, in some embodiments less than 10%, and insome embodiments less than 5%. In some embodiments, the phrase“substantially non-overlapping” as it relates to absorption spectrameans that the absorption spectra have peaks that differ by at least 15nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70nm, 75 nm, 80 nm, 85 nm, 90 nm, or 95 nm, all of which fall within therange of 650-1070 nm. Examples of absorption spectra that aresubstantially non-overlapping are those for B1, B2, and B3 in FIG. 1.

As used herein, the phrases “physiologically tolerable” and“pharmaceutically acceptable” refer to compositions, in some embodimentspharmaceutical compositions, that are recognized as being safe for usein a subject to which the compositions and methods of the presentlydisclosed subject matter are to be applied.

As used herein, the term “volume” refers to anything for which aphotoacoustic image might be desired. By way of example and notlimitation, a volume can be cell, tissue, or organ present in orisolated from a subject. In some embodiments, a volume can be aphysiologically relevant space or cavity within a subject.

II. Compositions of the Presently Disclosed Subject Matter

Unlike reagents derived from naturally occurring molecules such asbacteriochlorophyll a (see FIG. 2A), completely synthetic designs ofbacteriochlorins and bacteriochlorin derivatives provide access towavelength-tunable bacteriochlorins (Taniguchi et al., 2008). Anadvantage of being able to synthetically design bacteriochlorins andbacteriochlorin derivatives is that this facilitates development of apalette of NIR reagents with exceptionally narrow absorption bands thathave distinct benefits for PAI. Based on the designation offull-width-half-max (fwhm) to describe the width of a dye's absorption,a bacteriochlorin's NIR absorption band fwhm is typically less than orequal to 25 nm. Since the absorption maxima and other spectralproperties of synthetic bacteriochlorins can be readily “tuned”, aportfolio of matched bacteriochlorins and bacteriochlorin derivativescan be designed to fit within a smaller spectral range with minimaloverlap between the dyes. FIG. 1 illustrates the difference betweenabsorption spectra of three exemplary bacteriochlorins (B1-B3) and thatof ICG. Within the spectral space normally allotted to ICG detection,one could potentially detect up to three bacteriochlorins due to theirnarrow absorption profiles with minimal spectral overlap. Theseadvantages will allow more complex analyses of multiple disease-specificbiomarkers in the same experiment and ultimately accelerate thedevelopment of multiplex clinical assays based on PAI.

Bacteriochlorins are exemplified by bacteriochlorophyll a (see FIG. 2A),a natural product which is not readily amenable to extensive syntheticmodification because of the presence of numerous (hydrophobic)substituents and chiral centers. Its reduced pyrrole rings are alsosubject to dehydrogenation during synthetic manipulations. However, aseries of recent synthetic advances have provided access to stable,tailorable bacteriochlorins. The presence of a geminal dimethyl group inthe reduced pyrroline ring blocks dehydrogenation or oxidativeprocesses, affording a highly resilient bacteriochlorin (FIG. 2B). Byintroducing various groups at the beta-pyrrole positions (R2, R3, R12,R13 of FIG. 2B) and at meso-positions (R5, R15 of FIG. 2B) theabsorption maxima can be readily manipulated to provide desiredabsorptions anywhere from 700 nm to greater than 900 nm.

A second compelling advantage of bacteriochlorins for PAI is shown byresults described herein that demonstrate a substantial increase in PAIsignal by complexing bacteriochlorins with nickel. Free basebacteriochlorins and several metallobacteriochlorins (Zn, Pd) typicallydisplay fluorescence with quantum yields (Φ(f)) ranging up to 0.25, andin some cases substantial triplet states (up to 0.80 Φ(isc)). It isbelieved that the nickel-bacteriochlorin complex has an extremely rapidnon-radiative decay of its singlet state (ultrafast dynamics of B107suggest its decay to the ground state to be ˜10 ps) with essentially noconversion to its triplet state occurring. Without wanting to be boundby any specific mechanism of action, it is hypothesized that because theexcited state is so short-lived, this further enhances the PAI signal.Although these properties have been recognized previously and reportedin studies that characterized nickel derivatives of chlorophyll andbacteriochlorophyll (Pilch et al., 2013), attempts to produce andcharacterize PAI reagents with molecules of such a design has not beenreported. This is undoubtedly due to the aforementioned difficulty ofsynthetic manipulation of bacteriochlorophyll a and other naturalproducts. FIG. 3 shows PAI data for a nickel-bacteriochlorin (B107) incomparison to ICG using agar phantoms and a commercial PAI system with atunable laser scanning from 680-970 nm. It was determined that thiscompound displayed a five-fold stronger signal compared to ICG of anequivalent optical density at two depths of agar. This greatly enhancedsignal intensity suggested that PAI with targeted probes could be doneat substantially greater tissue depths than those currently accessiblewith ICG. The data is summarized in Table 1.

TABLE 1 PAI Signal Intensity at Indicated Wavelengths forNi-Bacteriochlorin and ICG Implanted in Agar Phantoms at DifferentSurface Depths Depth Dye Max (nm) Intensity 6 mm ICG 795 0.38 Ni-BC 7651.96 3 mm ICG 790 0.72 Ni-BC 760 3.50

A third advantage and further benefit of nickel-bacteriochlorincomplexes is their improved photostability. FIG. 4 shows the signals forICG and the nickel-bacteriochlorin B107 in agar phantoms measured withcontinuous 800 nm laser illumination. It is possible the enhancedstability may be due to the limited conversion to an excited tripletstate and subsequent limited degradation upon generation of singletoxygen.

In sum, some benefits of employing bacteriochlorins and bacteriochlorinderivatives as PAI contrast agents include the following:

Tunable wavelengths and narrow emissions will enable multiplexing,Synthetic design is amenable to adding solubilizing groups andbioconjugatable tethers;

Greatly enhanced signal metallobacteriochlorins (M═Ni, Fe, and/or Co)will enable PAI detection of biomarkers at greater depths thanconventional markers; and

Enhanced probe stability will be useful for photoacoustic microscopy,image-guided surgery and other procedures requiring extended imageacquisition times.

In addition to nickel bacteriochlorins, in some embodiments thepresently disclosed subject matter provides corresponding iron (Fe)and/or cobalt (Co) metallobacteriochlorins that provide enhancement ofsignal for PAI.

The metallobacteriochlorins can be used in some embodiments as contrastagents for general imaging of physiological features such as but notlimited to organs, veins, lymph nodes, and lymph systems, and in someembodiments they can be used as targeted probes by attaching targetingagents. As used herein, the phrase “targeting agent” refers to anymolecule that when attached to a composition of the presently disclosedsubject matter enhances the accumulation of the composition in a targetsite such as, but not limited to a cell, a tissue, or an organ. Forexample, attachment of a metallobacteriochlorin through a reactivelinker or tether to an antibody can be accomplished by methods whichhave been previously established for free base bacteriochlorins. In someembodiments, solubilizing groups such as carboxylates or PEG chains canimprove the biolabeling efficiency and bioconjugate stability of atargeting composition of the presently disclosed subject matter (i.e., abacteriochlorin or bacteriochlorin derivative to which a targeting agenthas been complexed). See e.g., Jiang et al., 2015; Zhang et al., 2016.

In some embodiments it can be preferred to incorporate ametallobacteriochlorin into a nanoparticle, microbead, micelle, or othercarrier structure to further enhance the PAI signal and/or to influencebiodistribution. Exemplary methods for incorporating hydroporphyrins,including bacteriochlorins and derivatives thereof such as but notlimited to metallobacteriochlorins, in microbeads are disclosed, forexample, in PCT International Patent Application Publication No. WO2017/214637, the entire content of which is incorporated herein byreference. Other nanoparticles include liposomes and doped silicananoparticles.

In addition to bacteriochlorins and derivatives thereof, in someembodiments other hydroporphyrins such as but not limited toisobacteriochlorins and some chlorins with longer wavelength NIRabsorptions are used for PAI panels.

Bacteriochlorins, metallobacteriochlorins, and their derivatives can insome embodiments be used in multi-color PAI panels as well asmulti-modal multi-color panels for imaging or image-guided therapy(e.g., image-guided surgery or image-guided drug delivery). Multi-modeexamples include fluorescence/PAI and MRI/PAI.

Non-limiting examples of water-soluble metallobacteriochlorins andmetallochlorins with tethers for bioconjugation are presented in FIG. 7.

III. Synthesis of Bacteriochlorins, Metallobacteriochlorins, andDerivatives Thereof

The bacteriochlorins that can serve as starting materials forsynthesizing the radiation-absorbing molecules of the presentlydisclosed subject matter can be produced by any method known to those ofskill in the art. Exemplary methods for synthesizing bacteriochlorinsand related molecules are disclosed in, for example, U.S. Pat. Nos.6,559,374; 7,470,785; 7,534,807; 8,129,520; 8,173,691; 8,173,692;8,207,329; 8,304,561; 8,664,260; 9,365,722; and 9,822,123; and in PCTInternational Patent Application Publication No. WO 2017/214637, thecontent of each of which is hereby incorporated by reference in itsentirety. Particular exemplary methods for synthesizing bacteriochlorinsand related molecules are as follows.

In some embodiments, a method for synthesizing a bacteriochlorin of thepresently disclosed subject matter comprises condensing a pair ofcompounds of Formula IIA:

in an organic solvent in the presence of an acid,

where each R′ independently represents C1-C4 alkyl, or both R′ togetherrepresent C2-C4 alkylene; to produce a compound of Formula I wherein R⁵is H or alkoxy;

when R⁵ is H, optionally brominating, and then optionally furthersubstituting the compound at the R⁵ position; to produce Formula IA,wherein Formula IA is:

wherein:

each R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is independently selected fromthe group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl, heterocyclo,heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, aryloxy,arylalkyl, arylalkenyl, arylalkynyl, heteroaryl, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, alkoxy, halo, mercapto, azido,cyano, formyl, carboxylic acid, hydroxyl, nitro, acyl, alkylthio, amino,alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy,ester, amide, sulfoxyl, sulfonyl, sulfonate, sulfonic acid, sulfonamide,urea, alkoxylacylamino, aminoacyloxy, hydrophilic groups, linkinggroups, surface attachment groups, and targeting groups;

or R¹ and R² together are ═O or spiroalkyl;

or R³ and R⁴ together are ═O or spiroalkyl;

or where R⁶ and R⁷, or R⁷ and R⁸, together represent a fused aromatic orheteroaromatic ring systems. In some embodiments, the compound ofFormula I can then be metalated, as desired.

Alternatively, a method for synthesizing a bacteriochlorin of thepresently disclosed subject matter can include condensing a compound ofFormula IIB and a compound of Formula III in a composition comprising afirst solvent to produce an intermediate;

wherein the compound of Formula IIB has a structure represented by:

wherein R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ are as provided below;

wherein the compound of Formula III has a structure represented by:

wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are as provided below; and

R²¹ and R²² are each independently selected from the group consisting ofhydrogen, alkyl and aryl, or R²¹ and R²² taken together represent aC2-C4 alkylene; and

condensing the intermediate in a second solvent in the presence of anacid to produce the compound of Formula IV or a metal conjugate thereof,wherein Formula IV is defined as:

or a metal conjugate thereof (e.g., a metal chelate thereof), wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, andR¹⁶ are each independently selected from the group consisting of H,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl,cycloalkylalkynyl, heterocyclo, heterocycloalkyl, heterocycloalkenyl,heterocycloalkynyl, aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl,heteroaryl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,alkoxy, halo, mercapto, azido, cyano, formyl, carboxylic acid, hydroxyl,nitro, acyl, alkylthio, amino, alkylamino, arylalkylamino, disubstitutedamino, acylamino, acyloxy, ester, amide, sulfoxyl, sulfonyl, sulfonate,sulfonic acid, sulfonamide, urea, alkoxylacylamino, aminoacyloxy,hydrophilic groups, linking groups, surface attachment groups, andtargeting groups;

or R¹ and R² together are ═O or spiroalkyl;

or R³ and R⁴ together are ═O or spiroalkyl;

or R⁹ and R¹⁰ together are ═O or spiroalkyl;

or R¹¹ and R¹² together are ═O or spiroalkyl;

or R¹⁵ and R¹⁶ together are ═O;

or R⁵ and R⁶ together represent a fused aromatic or heteroaromatic ringsystems;

or R⁶ and R⁷ together represent a fused aromatic or heteroaromatic ringsystems;

or R¹³ and R¹⁴ together represent a fused aromatic or heteroaromaticring systems; and

Z is an electron-withdrawing group (e.g., —CO₂R¹⁷, —C(O)NHR¹⁷,—C(O)NR¹⁷R¹⁸, —C(O)R¹⁷, —CN, —C═N—NR¹⁷R¹⁸, —PO(OR¹⁷)₂, —SO₂OR¹⁷,—SO₂NR¹⁷R¹⁸, —SO₂R¹⁷, and —SiR¹⁷R¹⁸R¹⁹, and wherein R¹⁷, R¹⁸, and R¹⁹are, in each occurrence, independently selected from the groupconsisting of hydrogen, alkyl and aryl).

Additional exemplary routes to synthesis of bacteriochlorins include theNorthern-Southern Route described in Liu & Lindsey, 2016 and the methodsfor synthesizing bacteriochlorin macrocycles with annulated isocyclicrings described in Zhang & Lindsey, 2017, the content of each of whichis incorporated herein by reference in its entirety.

Three exemplary bacteriochlorins that can be employed in abacteriochlorin panel (e.g., bacteriochlorins B1-B3) are shown in FIG.5. These can be converted to the corresponding metallobacteriochlorins(depicted in FIG. 8) which would be expected to have a similar spread ofpeak signal in PAI with low overlap between them and a 20-30 nm shift inpeak spectral wavelengths from the free base bacteriochlorins. FIG. 6 isa brief outline of methods of synthesis of bacteriochlorins B1-B3.Bacteriochlorins B1 and B2 can be synthesized by known methods (seeKrayer et al., 2010) in five steps, starting from the correspondingpyrole-2-carboxaldehyde. Bacteriochlorin B3 requires thebromo-substituted bacteriochlorin (R¹═Br) undergoing Sonogashiracoupling to install the phenylethynyl groups to achieve the desiredabsorption at ˜790 nm. An analogue of bacteriochlorin B3 has beensynthesized and was verified to meet this wavelength expectation.

Metalation of each bacteriochlorin can be achieved by recently developedmethods as essentially described in Chen et al., 2012. The metalation ofsynthetic bacteriochlorins was advanced to cover the syntheticbacteriochlorins bearing various substitution patterns, ranging fromelectron-withdrawing to electron-rich functions. In general, two methodscan be utilized in the metalation depending on the nature of thebacteriochlorins. The electron-rich bacteriochlorins can be metalated bytreating with strong bases (NaH or LDA) in THF, following by addition ofmetal salts (MX_(n)) at 60° C. The electron-deficient bacteriochlorinscan be metalated by treating with metal salts (MX_(n)) in DMF atelevated temperature. The two methods have been previously used toprepare various synthetic Zn—, Cu—, Pd— and Ni-bacteriochlorins. Thesemethods can also be employed for synthetic Fe— and Co-bacteriochlorinsvia metalation of free-base bacteriochlorins with Fe and Co.

IV. Methods of PAI using the Compositions of the Presently DisclosedSubject Matter

Photoacoustic imaging is a technique wherein non-ionizing laser pulsesare delivered to biological tissues. A fraction of the delivered energyis absorbed and converted into heat, leading to transient thermoelasticexpansion and ultrasonic emission. The generated ultrasonic waves arethereafter detected and analyzed to produce images of the biologicaltissues. Generally, the magnitude of the ultrasonic emission revealsphysiologically specific optical absorption contrast. 2D or 3D images ofthe targeted areas can then be formed. See e.g., U.S. Patent ApplicationPublication Nos. 2005/0085725; 2009/0066949; 2009/0069653; 2010/0226003;and 2012/0296192; U.S. Pat. Nos. 6,738,653; 7,864,307; 7,916,283; PCTInternational Patent Application Publication No. WO 2002/008740; and Xu& Wang, 2006; Li & Wang, 2009; Li et al., 2009; Wang, 2009; Yang et al.,2009; each of which is incorporated herein by reference in its entirety.

As such, in some embodiments the presently disclosed subject matterprovides methods for generating an image of a volume. In someembodiments, the methods comprise (a) contacting the volume with acontrast agent comprising at least one radiation-absorbing componentcomprising a bacteriochlorin, a metallobacteriochlorin, a derivativethereof, or a combination thereof; (b) exposing the volume to radiation;(c) detecting ultrasonic waves generated in the volume by the radiation;and (d) generating a photoacoustic image therefrom of the volume or partthereof containing the contrast agent.

The presently disclosed subject matter also provides in some embodimentsmethods for multiplex photoacoustic imaging of a volume. In someembodiments, the methods comprise (a) contacting the volume with acontrast agent comprising a plurality of radiation-absorbing components,each member of the plurality of radiation-absorbing componentscomprising a bacteriochlorin, a metallobacteriochlorin, a derivativethereof, or a combination thereof; (b) exposing the volume to radiation,wherein the radiation is calibrated to wavelengths that aredifferentially absorbed by the plurality of radiation-absorbingcomponents; (c) differentially detecting ultrasonic waves generated inthe volume by the radiation as it is differentially absorbed by theplurality of radiation-absorbing components; and (d) generating aphotoacoustic image therefrom of the volume or a part thereof containingthe administered contrast agent, wherein the photoacoustic image isgenerated from the differentially detecting ultrasonic waves.

Thus, the presently disclosed methods can be employed in in vivo, exvivo, and in vitro uses. When employed in vivo, the presently disclosedmethods can employ contrast agents that are physiologically tolerablefor use in a subject, optionally a human. In some embodiments, thecontrast agents are formulated as part of a pharmaceutical composition,which in some embodiments can further comprise one or morepharmaceutically acceptable carriers, diluents, and/or excipient. Insome embodiments, the pharmaceutical composition is pharmaceuticallyacceptable for use in a human. Suitable formulations include aqueous andnon-aqueous sterile injection solutions which can contain anti-oxidants,buffers, bacteriostats, bactericidal antibiotics, and solutes whichrender the formulation isotonic with the bodily fluids of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which caninclude suspending agents and thickening agents. The formulations can bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and can be stored in a frozen or freeze-dried(lyophilized) condition requiring only the addition of sterile liquidcarrier, for example water for injections, immediately prior to use.Some exemplary ingredients are sodium dodecyl sulfate (SDS) in the rangeof in some embodiments 0.1 to 10 mg/ml, in some embodiments about 2.0mg/ml; and/or mannitol or another sugar in the range of in someembodiments 10 to 100 mg/ml, in some embodiments about 30 mg/ml; and/orphosphate-buffered saline (PBS). Any other agents conventional in theart having regard to the type of formulation in question can be used.

In some embodiments, the presently disclosed compositions are employedas contrast agents and/or as components of multi-color PAI panels and/ormulti-modal multi-color panels for imaging or image-guided therapy (seee.g., U.S. Pat. No. 8,617,522; incorporated herein by reference).

EXAMPLES

The following Examples provide illustrative embodiments. In light of thepresent disclosure and the general level of skill in the art, those ofskill will appreciate that the following Examples are intended to beexemplary only and that numerous changes, modifications, and alterationscan be employed without departing from the scope of the presentlydisclosed subject matter.

Materials and Methods used in the EXAMPLES

Indocyanine Green (ICG), was obtained from Sigma-Aldrich (Catalogue No.12633; Sigma-Aldrich Corp., St. Louis, Mo., United States of America).The nickel bacteriochlorin B107 was prepared as described in Sun et al.,2013 and Chen et al, 2012. Each dye was dissolved inN,N-dimethylformamide (DMF) at the desired concentrations prior to theimaging experiments. Molds for preparing agar phantoms were typically 90mm diameter glass Petri dishes.

Example 1

Phantom Preparation

A highly purified agar powder (Catalogue No. A7921; Sigma-Aldrich Corp.,St. Louis, Mo., United States of America) was dissolved in water(Reagent Grade, Type I) to a final concentration of 2.0% and heated tothe melting temperature of 95° C. in a microwave oven. Three 30 secondcycles of heating and swirling to mix resulted in a smooth agarpreparation. The bottle containing agar was held at 75-85° C. for 1-3hours using a standard hot plate and a double boiler to avoid scorching(which causes a detectable increase in the absorption coefficient). Thiswaiting period at an elevated temperature allows the slow release of airbubbles and produces an agar solution with negligible absorption andvery low turbidity. The desired optical properties of the phantom werereached by adding a 20% (1:5 v/v) final concentration of 1.0% low fatmilk as a scattering medium and India ink (Higgins Black 44201;Chartpak, Inc., Leeds, Mass., United States of America) as an absorbingmedium. These additions were made in the range of 54-58° C. to avoidprecipitation of the milk proteins. The solution was stirred slowly andcontinuously with a stir bar at a speed to maintain homogeneity of themilk and ink, but not cause bubbles or foaming.

Petri dishes were pre-warmed on a 50° C. hot plate for 1 minute prior tothe dispensing of the Phantom matrix The phantom matrix solution wasdispensed into the molds using a pre-warmed 25 ml serological pipetteand left undisturbed to reach proper hardening and stable opticalproperties (20-25° C.). If the formed Phantoms were not used within fourhours, they were sealed to limit evaporation. In general, the formedPhantoms were used within 8 hours or within 72 hours if storedrefrigerated. If refrigerated, they were warmed to ambient temperatureprior to use. This allows the phantom matrix to spread evenly prior tohardening allowing for optical flatness of the phantom surface (quicklyaspirated and dispensed two times in the 50-53° C. Phantom matrix).Typically, 20 ml of the phantom matrix was added to obtain 3±10% mmthick molds. The 3 mm thick phantoms were stacked as needed in order tomeasure the PAI signal from dyes in polyethylene tubing extended acrossand under the agar layers at the desired depth.

Example 2

Photoacoustic Imaging of Agar Phantoms and Dyes

Photoacoustic imaging was performed using a VEVO® LAZR 2100 imagingsystem (VisualSonics, Inc., Toronto, Ontario, Canada) equipped withsoftware version 1.7.2. This instrument combines Ultrasound withPhotoacoustics Mode (PA) imaging and employs an optical parametricoscillator laser (OPO) pumped by a doubled Nd:YAG tunable laser. Imagingwas performed in PA scanning mode (680-900 nm) with a 5 nm step size orat fixed wavelengths for a designated period of time. Dyes wereintroduced via syringe into PESO polyethylene tubing (0.023″×0.038″;Braintree Scientific, Braintree, Mass., United States of America) andclamped at each end during the imaging experiments. Ultrasound gel wasapplied to the surface of the agar phantoms to ensure efficient couplingbetween the transducer and the Phantoms. The tubing and its associatedimage regions were isolated by ultrasound and PA signals for PAintensity quantitation. The data presented in FIGS. 3 and 4 were forsolutions of 63 μM Ni-bacteriochlorin (B107) and 50 μM ICG, andindicated an approximately 5-fold greater signal for theNi-bacteriochlorin at two depths of agar compared to ICG.

Example 3

Preparation of other Metallobacteriochlorins, and Photoacoustic Imagingof Agar Phantoms and Dyes using the Same

Cobalt- (Co) and Iron- (Fe) bacteriochlorins that correspond toNi-bacteriochlorin B107 are also prepared using the basic schemedepicted in FIG. 6. Additionally, Ni—, Co—, and Fe-bacteriochlorins thatcorrespond to bacteriochlorins B1-B3 are also prepared using the basicscheme depicted in FIG. 6. Exemplary metalated bacteriochlorins arepresented in FIG. 8.

Photoacoustic imaging of agar phantoms and dyes using the Co— and Fe—bacteriochlorin derivatives of B107 and the Ni—, Co—, andFe-bacteriochlorin derivatives of bacteriochlorins B1-B3 are performedessentially as set forth in EXAMPLE 2. The intensities of the varioussignals and the normalized signals are compared to each other and tothose of ICG.

REFERENCES

All references listed below, as well as all references cited in theinstant disclosure, including but not limited to all patents, patentapplications and publications thereof, scientific journal articles, anddatabase entries are incorporated herein by reference in theirentireties to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, and/or compositionsemployed herein.

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It will be understood that various details of the presently disclosedsubject matter may be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

What is claimed is:
 1. A photoacoustic imaging contrast agent comprisingat least one radiation-absorbing component comprising a bacteriochlorin,a metallobacteriochlorin, a derivative thereof, or a combinationthereof.
 2. The photoacoustic imaging contrast agent of claim 1,comprising a plurality of different bacteriochlorins,metallobacteriochlorins, derivatives thereof, or combinations thereof,each bacteriochlorin, metallobacteriochlorin, or derivative having adifferent absorption spectrum in the range of 650-1070 nm.
 3. Thephotoacoustic imaging contrast agent of claim 2, wherein: thephotoacoustic imaging contrast agent comprises at least three differentbacteriochlorins, metallobacteriochlorins, or derivatives thereof; (ii)each bacteriochlorin, metallobacteriochlorin, or derivative thereof hasan absorption spectrum with a peak absorption value in the range of700-950 nm; and (iii) the at least three absorption spectra aresubstantially non-overlapping in the range of 700-950 nm.
 4. Thephotoacoustic imaging contrast agent of claim 1, wherein themetallobacteriochlorin comprises a metal selected from the groupconsisting of nickel, iron, and cobalt.
 5. A method of generating animage of a volume, the method comprising: (a) contacting the volume witha contrast agent comprising at least one radiation-absorbing componentcomprising a bacteriochlorin, a metallobacteriochlorin, a derivativethereof, or a combination thereof; (b) exposing the volume to radiation;(c) detecting ultrasonic waves generated in the volume by the radiation;and (d) generating a photoacoustic image therefrom of the volume or partthereof containing the contrast agent.
 6. The method of claim 5, whereinthe bacteriochlorin, the metallobacteriochlorin, or the derivativethereof is a component of and/or encapsulated in a micelle, a liposome,a nanoparticle, or a combination thereof.
 7. The method of claim 5,wherein radiation with a wavelength of 650-1070 nm is used.
 8. Themethod of claim 7, wherein radiation with a wavelength of 650-900 nm,700-950 nm, and/or 750-950 nm is used.
 9. The method of claim 5, whereinthe contrast agent comprises a plurality of different bacteriochlorins,metallobacteriochlorins, derivatives thereof, or combinations thereof,each bacteriochlorin, metallobacteriochlorin, or derivative having adifferent absorption spectrum in the range of 650-1070 nm.
 10. Themethod of claim 5, wherein the contrast agent comprises a targetingagent.
 11. The method of claim 10, wherein the targeting agent comprisesa moiety that binds to a ligand and/or a target present on a tumor cellor a cancer cell, or a vascular endothelial cell associated therewith.12. The method of claim 11, wherein the ligand and/or a target comprisesa tumor-associated antigen.
 13. The method of claim 11, wherein themoiety comprises a peptide or peptide mimetic that binds to thetumor-associated antigen.
 14. A method for multiplex photoacousticimaging of a volume, the method comprising: (a) contacting the volumewith a contrast agent comprising a plurality of radiation-absorbingcomponents, each member of the plurality of radiation-absorbingcomponents comprising a bacteriochlorin, a metallobacteriochlorin, aderivative thereof, or a combination thereof; (b) exposing the volume toradiation, wherein the radiation is calibrated to wavelengths that aredifferentially absorbed by the plurality of radiation-absorbingcomponents; (c) differentially detecting ultrasonic waves generated inthe volume by the radiation as it is differentially absorbed by theplurality of radiation-absorbing components; and (d) generating aphotoacoustic image therefrom of the volume or a part thereof containingthe administered contrast agent, wherein the photoacoustic image isgenerated from the differentially detecting ultrasonic waves.
 15. Themethod of claim 14, wherein one or more of the plurality of thebacteriochlorins, metallobacteriochlorins, or derivatives thereof is acomponent of and/or encapsulated in a micelle, a liposome, ananoparticle, or a combination thereof.
 16. The method of claim 14,wherein radiation with a wavelength of 650-1070 nm is used.
 17. Themethod of claim 16, wherein radiation with a wavelength of 650-900 nm,700-950 nm, and/or 750-950 nm is used.
 18. The method of claim 14,wherein each member of the plurality of radiation-absorbing componentshas a different absorption spectrum in the range of 650-1070 nm.
 19. Themethod of claim 14, wherein one or more of the members of the pluralityof radiation-absorbing components comprises a targeting agent.
 20. Themethod of claim 19, wherein the targeting agent comprises a moiety thatbinds to a ligand and/or a target present on a tumor cell or a cancercell, or a vascular endothelial cell associated therewith.
 21. Themethod of claim 20, wherein the ligand and/or a target comprises atumor-associated antigen.
 22. The method of claim 20, wherein the moietycomprises a peptide or peptide mimetic that binds to a tumor-associatedantigen.
 23. The method of claim 14, wherein two or more of the membersof the plurality of radiation-absorbing components comprise a targetingagent.
 24. The method of claim 23, wherein the two or more of themembers of the plurality of radiation-absorbing components comprisedifferent targeting agents.
 25. The method of any one of claims 5-24,wherein the volume is a subject or a body part thereof, optionally acell, tissue, and/or organ thereof.
 26. The method of claim 27, whereinthe volume comprises a tumor cell, a cancer cell, or a tumor- orcancer-associated vascular cell.
 27. The method of any one of claims5-26, wherein the contrast agent is physiologically tolerable for use ina subject, optionally a human.
 28. The method of any one of claims 5-27,wherein the contrast agent is provided in a pharmaceutical compositioncomprising the photoacoustic imaging contrast agent and apharmaceutically acceptable carrier, diluent, or excipient.
 29. Themethod of claim 28, wherein the pharmaceutical composition ispharmaceutically acceptable for use in a human.
 30. A photoacousticimaging contrast agent comprising at least one radiation-absorbingcomponent comprising a bacteriochlorin, a metallobacteriochlorin, aderivative thereof, or a combination thereof, wherein the at least oneradiation-absorbing component comprises a compound selected from thegroup consisting of:

or a metalized version thereof.
 31. The photoacoustic imaging contrastagent of claim 30, wherein the at least one radiation-absorbingcomponent comprises a metalized derivative of B1-B3 and B107 comprisinga complexed metal, wherein the complexed metal is selected from thegroup consisting of nickel, cobalt, and iron.
 32. The photoacousticimaging contrast agent of any one of claims 1-4, 30, and 31, wherein thephotoacoustic imaging contrast agent is physiologically tolerable foruse in a subject, optionally a human.
 33. A pharmaceutical compositioncomprising the photoacoustic imaging contrast agent of any one of claims1-4 and 30-32 and a pharmaceutically acceptable carrier, diluent, orexcipient.
 34. The pharmaceutical composition of claim 33, wherein thepharmaceutical composition is pharmaceutically acceptable for use in ahuman.